RECEPTOR-MEDIATED ACTIVATION OF G(S-ALPHA) - EVIDENCE FOR INTRAMOLECULAR SIGNAL-TRANSDUCTION

To investigate the mechanism by which cell surface receptors activate heterotrimeric G proteins, we applied a scanning mutagenesis approach to the carboxyl-terminal 40% of alpha(s) (residues 236-394) to identif y residues that play a role in receptor-mediated activation. We identi fied four regions of sequence in which mutations significantly impaire d receptor-dependent stimulation of cAMP synthesis in transiently tran sfected cyc(-) S49 lymphoma cells, which lack endogenous alpha(s). Res idues at the carboxyl terminus are likely to be receptor contact sites . Buried residues near the bound GDP are connected to the carboxyl ter minus by an a helix and may regulate GDP affinity. Residues in two adj acent loops of the GTPase domain at the interface with the helical dom ain, one of which includes a region, switch III, that changes conforma tion on GTP binding, are positioned to relay the receptor-initiated si gnal across the domain interface to facilitate GDP release. Consistent with this hypothesis, replacing the helical domain of alpha(s) with t hat of alpha(i2) in an alpha(s)/alpha(i2)/alpha(s) chimera corrects th e defect in receptor-mediated activation caused by alpha(i2) substitut ions on the GTPase side of the interface. Thus, complementary interact ions between residues across the domain interface seem to play a role in receptor-catalyzed activation.